JP7447810B2 - Method for producing tripeptide γ-GLU-VAL-GLY using Enterobacteriaceae - Google Patents

Description

The present invention relates to microbial industry, in particular to a method for producing tripeptides, and more particularly to tripeptide γ-Glu-Val-Gly. The method of the invention uses bacteria of the family Enterobacteriaceae that have been modified to overexpress at least the tdh and kbl genes.

Methods for producing amino acids and peptides by fermentation using mutant microorganisms or microorganisms resistant to various drugs have been reported so far. Conventional methods for producing such mutant strains include subjecting microorganisms to mutation treatments such as ultraviolet irradiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), and then subjecting them to appropriate treatments. There is a method of selecting a desired strain using a selective medium. Alternatively, mutant strains can be bred using genetic engineering techniques that involve overexpressing genes involved in desired amino acid and peptide biosynthetic pathways.

Production of tripeptides such as γ-Glu-Val-Gly has conventionally been achieved by chemical and enzymatic methods rather than by utilizing microbial fermentation. For example, WO 2015133547 A1 describes a multi-step method for producing γ-Glu-Val using various synthetic enzymes and then producing γ-Glu-Val-Gly. WO 2014025023 A1 describes a method for producing γ-Glu-Val-Gly crystals using basic enzymatic and chemical methods. However, the utilization of fermentation of microorganisms designed to produce γ-Glu-Val-Gly has not been described so far.

γ-Glu-Val-Gly is known to be useful in both the food and flavor industries. For example, this tripeptide has been reported to have excellent kokumi quality and is therefore used in flavored teas (WO 2015136841 A1), alcoholic beverages (Miyamura et al., J. Biosci. and Bioeng. (2015) ) 120(3):311-314), seasonings (Miyamura et al., Food Sci. and Technol. Res. (2014) 20(3):699-703), and spices (WO 2014123175 A1). ing. Therefore, efficient and successful overproduction of the tripeptide γ-Glu-Val-Gly by microbial fermentation is highly desirable.

L-threonine 3-dehydrogenase and/or 2-amino-3-oxobutanoic acid coenzyme A ligase in the production of the tripeptide γ-Glu-Val-Gly by fermentation of Enterobacteriaceae bacteria. The effects of overexpression of the gene encoding (2-amino-3-oxobutanoate coenzyme A ligase) have not been reported so far.

One aspect of the present invention is a method for producing γ-Glu-Val-Gly, comprising:
(i) Cultivate γ-Glu-Val-Gly-producing bacteria belonging to the Enterobacteriaceae family in a culture medium to inject γ-Glu-Val-Gly into the culture medium, the bacterial cells, or both. and (ii) recovering γ-Glu-Val-Gly from the culture medium or the bacterial cells, or both;
The method, wherein the bacterium is modified to overexpress a gene encoding a protein having L-threonine 3-dehydrogenase activity and a gene encoding a protein having 2-amino-3-oxobutanoic acid coenzyme A ligase activity. The goal is to provide the following.

A further aspect of the present invention is that the protein having L-threonine 3-dehydrogenase activity is encoded by the tdh gene, and the protein having 2-amino-3-oxobutanoic acid coenzyme A ligase activity is encoded by the kbl gene. , to provide the method.
A further aspect of the invention is the method, comprising:
The protein having L-threonine 3-dehydrogenase activity is selected from the group consisting of:
A protein comprising the amino acid sequence shown in SEQ ID NO: 2;
A protein having L-threonine 3-dehydrogenase activity, comprising an amino acid sequence containing substitutions, deletions, insertions, and/or additions of about 1 to 50 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2; and A protein comprising an amino acid sequence having 60% or more identity to the entire amino acid sequence shown in number 2 and having L-threonine 3-dehydrogenase activity;
and,
The method, wherein the protein having 2-amino-3-oxobutanoic acid coenzyme A ligase activity is selected from the group consisting of:
A protein comprising the amino acid sequence shown in SEQ ID NO: 4;
The amino acid sequence shown in SEQ ID NO: 4 contains an amino acid sequence containing substitutions, deletions, insertions, and/or additions of about 1 to 50 amino acid residues, and has 2-amino-3-oxobutanoic acid coenzyme A ligase activity. and a protein comprising an amino acid sequence having 60% or more identity to the entire amino acid sequence shown in SEQ ID NO: 4, and having 2-amino-3-oxobutanoic acid coenzyme A ligase activity.

A further aspect of the invention is the method, comprising:
The protein having L-threonine 3-dehydrogenase activity is encoded by a DNA selected from the group consisting of:
DNA containing the base sequence shown in SEQ ID NO: 1;
DNA containing a nucleotide sequence that is complementary to the sequence shown in SEQ ID NO: 1 and a nucleotide sequence that can hybridize under stringent conditions;
A DNA encoding a protein comprising an amino acid sequence containing substitutions, deletions, insertions, and/or additions of about 1 to 50 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2,
DNA in which the protein has L-threonine 3-dehydrogenase activity; and DNA that is a variant base sequence of SEQ ID NO: 1 due to degeneracy of the genetic code;
and,
A method, wherein the protein having 2-amino-3-oxobutanoic acid coenzyme A ligase activity is encoded by a DNA selected from the group consisting of:
DNA containing the base sequence shown in SEQ ID NO: 3;
DNA containing a nucleotide sequence that is complementary to the sequence shown in SEQ ID NO: 3 and a nucleotide sequence that can hybridize under stringent conditions;
A DNA encoding a protein comprising an amino acid sequence containing substitutions, deletions, insertions, and/or additions of about 1 to 50 amino acid residues in the amino acid sequence shown in SEQ ID NO: 4,
DNA in which the protein has 2-amino-3-oxobutanoic acid coenzyme A ligase activity; and DNA that is a mutant base sequence of SEQ ID NO: 3 due to degeneracy of the genetic code.
The goal is to provide the following.

A further aspect of the present invention is that the gene encoding a protein having L-threonine 3-dehydrogenase activity and the gene encoding a protein having 2-amino-3-oxobutanoic acid coenzyme A ligase activity each consist of the following: and the expression of those genes is increased compared to unmodified bacteria:
increasing the copy number of the gene or those genes in the bacterium;
modifying the gene or the expression control region of those genes in the bacterium; and a combination thereof.

A further aspect of the invention is to provide said method, wherein said bacterium is further modified by a method selected from the group consisting of:
Attenuating gcvP gene expression;
Attenuating the expression of the sucAB operon gene;
Overexpressing the tolC gene;
overexpressing the ilvGMEDA operon genes; and combinations thereof.

A further aspect of the invention is to provide the method, wherein the ilvGMEDA operon gene comprises the ilvG gene encoding acetolactate synthase II, and the activity of the acetolactate synthase II is restored.

A further aspect of the invention is to provide the method, wherein the bacterium belongs to the genus Escherichia or Pantoea.

A further aspect of the invention is to provide the method, wherein the bacterium is Escherichia coli or Pantoea ananatis.
Further objects, features, and attendant advantages of the present invention will become apparent to those skilled in the art from reading the following detailed description of the embodiments.

1. Bacteria In the methods described herein, a gene encoding a protein with L-threonine 3-dehydrogenase activity and 2-amino-3-oxobutanoic acid coenzyme A ligase (2-amino- Any γ-Glu-Val-Gly producing bacterium that has been modified to overexpress both genes encoding proteins with 3-oxobutanoate coenzyme A ligase activity can be used.

The bacteria to be modified (i.e., the bacteria before the introduction of the modification that overexpresses both genes) has a gene encoding a protein with L-threonine 3-dehydrogenase activity and a 2-amino-3-oxobutanoate coenzyme A ligase activity. Bacteria that can be modified to overexpress both protein-encoding genes (i.e., bacteria after introduction of the modification that overexpress both genes) contain the tripeptide γ-Glu-Val- There are no particular restrictions as long as Gly can be produced. Examples of bacteria will be described later.

The term "the bacterium is capable of producing the tripeptide γ-Glu-Val-Gly" refers to the bacteria after the introduction of the modification. However, this may mean that the ability to produce the tripeptide γ-Glu-Val-Gly was obtained by introducing the modification. In addition, the term "bacteria can produce the tripeptide γ-Glu-Val-Gly" refers to bacteria after the introduction of the modification. It can mean that it has become like this.

The term "γ-Glu-Val-Gly-producing bacteria" is the term for "bacteria capable of producing the tripeptide γ-Glu-Val-Gly" or "bacteria capable of producing the tripeptide γ-Glu-Val-Gly". can be equivalent to the term The term "γ-Glu-Val-Gly producing bacterium" may mean a bacterium capable of producing the tripeptide γ-Glu-Val-Gly by fermentation of the bacterium in a culture medium. In addition, the term "γ-Glu-Val-Gly-producing bacteria" means that when the bacteria is cultured in a culture medium, it produces and excretes the tripeptide γ-Glu-Val-Gly in the medium and/or the bacterial body. Alternatively, it can mean bacteria that can secrete and/or accumulate. Specifically, the term "γ-Glu-Val-Gly-producing bacteria" refers to the ability to recover the tripeptide γ-Glu-Val-Gly from the culture medium and/or bacterial cells when the bacteria is cultured in a culture medium. To some extent, it can refer to bacteria that can produce, excrete or secrete, and/or accumulate the tripeptide γ-Glu-Val-Gly in the medium and/or the bacterial body. "Bacteria are cultivated in a medium"
can be equivalent to the term "bacteria are cultured in a medium", and these terms are known to those skilled in the art. Furthermore, the term "γ-Glu-Val-Gly-producing bacteria" specifically refers to non-modified strains, such as Escherichia coli (E. coli) K-12 (including E. coli K-12 MG1655), etc. can refer to a bacterium that is capable of producing, excreting or secreting, and/or accumulating the tripeptide γ-Glu-Val-Gly in larger amounts in the medium compared to the wild-type or parent strain of . In addition, the term "γ-Glu-Val-Gly-producing bacteria" specifically refers to the tripeptide γ-Glu-Val-Gly in an amount of 0.01 g/L or more, for example, 0.1 g/L or more, 0.5 g It can mean a bacterium that can produce and accumulate the tripeptide γ-Glu-Val-Gly in an amount of 1.0 g/l or more, or 1.0 g/l or more in a medium and/or a bacterial cell. The term "γ-Glu-Val-Gly-producing bacteria" specifically refers to the tripeptide γ-Glu-Val-Gly in an amount of 0.01 g/L or more, such as 0.1 g/L or more, 0.5 g/L or more, or It can mean a bacterium that can produce and accumulate the tripeptide γ-Glu-Val-Gly in an amount of 1.0 g/l or more.

The term “tripeptide γ-Glu-Val-Gly” (which may be used interchangeably or equivalently with the term “γ-Glu-Val-Gly” (also abbreviated as “γ-EVG”)) A tripeptide as described herein may refer to a peptide (a peptide comprising three amino acid residues covalently linked to each other in a chain), wherein the tripeptide described herein comprises a glycine (Gly) residue; The amino group of is bonded to the carboxyl group of valine (Val) residue, and the amino group of Val residue is bonded to glutamic acid (Glu).
It is attached to the carboxyl group of the residue through the γ-carbon atom (PubChem CID: 25099093).

Bacteria can produce the tripeptide γ-Glu-Val-Gly in free form, or its salts or hydrates, or its adducts (i.e., formed by γ-Glu-Val-Gly and another organic or inorganic compound). adducts) or mixtures thereof. Therefore, the term "γ-Glu-Val-Gly" is not limited to the free form of γ-Glu-Val-Gly, but also includes salts or hydrates of γ-Glu-Val-Gly, or adducts thereof (i.e. adducts formed by γ-Glu-Val-Gly and another organic or inorganic compound) may also be included. That is, the term "γ-Glu-Val-Gly" can mean γ-Glu-Val-Gly in free form, a salt or hydrate thereof, an adduct thereof, or a mixture thereof. The term "γ-Glu-Val-Gly" may particularly mean γ-Glu-Val-Gly in free form, its salts, or mixtures thereof. It is also accepted that the bacteria may produce γ-Glu-Val-Gly alone or as a mixture of γ-Glu-Val-Gly and one or more other amino acids or peptides. The term "L-amino acid" includes L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L- L-forms such as isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, etc. of the amino acid (also referred to as the L-enantiomer of the amino acid). The term "peptide" may include dipeptides such as γ-Glu-Val and Val-Gly.

Bacteria may be naturally capable of producing the tripeptide γ-Glu-Val-Gly, or may be modified to be capable of producing the tripeptide γ-Glu-Val-Gly. Such modifications can be achieved, for example, by mutagenesis or DNA recombination techniques. That is, bacteria can produce L-threonine in bacteria that can naturally produce the tripeptide γ-Glu-Val-Gly or in bacteria that have been modified to be able to produce the tripeptide γ-Glu-Val-Gly. It can be obtained by overexpressing a gene encoding a protein having 3-dehydrogenase activity and a gene encoding a protein having 2-amino-3-oxobutanoate coenzyme A ligase activity. Alternatively, the bacterium can contain a gene encoding a protein with L-threonine 3-dehydrogenase activity and 2-amino-3-oxobutanoate coenzyme A ligase, allowing the bacterium to produce the tripeptide γ-Glu-Val-Gly. It can be obtained by overexpressing a gene encoding an active protein. That is, the bacterium has a gene encoding a protein with L-threonine 3-dehydrogenase activity and a 2-amino-3-oxobutanoate coenzyme A ligase so that the modified bacterium can produce the tripeptide γ-Glu-Val-Gly. It can be modified to overexpress genes encoding active proteins.

The bacteria described herein can be, for example, Gram-negative bacteria, and specific examples of Gram-negative bacteria include bacteria belonging to the family Enterobacteriaceae. The following description of bacteria is applicable mutatis mutandis to any bacteria used in the methods described herein.

In the methods described herein, the bacteria belonging to the family Enterobacteriaceae can be bacteria belonging to the genera Enterobacter, Erwinia, Escherichia, Klebsiella, Morganella, Pantoea, Photorhabdus, Providencia, Salmonella, Yersinia, etc. , and can produce the tripeptide γ-Glu-Val-Gly. Specifically, it is classified into Enterobacteriaceae according to the classification method used in the NCBI (National Center for Biotechnology Information) database (ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=543). Bacteria that have been used can be used. Examples of Enterobacteriaceae bacteria that can be modified include Escherichia
, Enterobacter, or Pantoea.

Strains of the Escherichia genus bacteria that can be modified to obtain the Escherichia genus bacteria described herein are not particularly limited; mutant derivatives of Escherichia coli K-12, p. 2460-2488. In FC Neidhardt et al. (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2 ^nd ed. ASM Press, Washington, DC, 1996). Those listed can be used. In particular, Escherichia coli (E. coli) may be mentioned. Specific examples of E. coli include E. coli W3110 (ATCC 27325) and E. coli MG1655 (ATCC 47076) derived from E. coli K-12 strain, which is a prototype wild strain. These strains can be obtained, for example, from the American Type Culture Collection (ATCC), as described above. Examples of bacteria of the genus Enterobacter include Enterobacter agglomerans and Enterobacter aerogenes, and examples of bacteria of the genus Pantoea include Pantoea ananatis. In recent years, some strains of Enterobacter agglomerans have been reclassified as Pantoea agglomerans, Pantoea ananatis, or Pantoea stewartii based on 16S rRNA sequence analysis. ing. Bacteria classified into the Enterobacteriaceae family can be used regardless of whether they belong to the genus Enterobacter or the genus Pantoea. When breeding P. ananatis strains using genetic engineering methods, P. ananatis strains AJ13355 (FERM BP-6614), AJ13356 strains (FERM BP-6615), AJ13601 strains (FERM BP-7207), and Derivative strains of can be used. When these strains were isolated, they were identified as Enterobacter agglomerans and deposited as Enterobacter agglomerans, but as mentioned above, they have recently been reclassified as P. ananatis based on 16S rRNA nucleotide sequence analysis and other factors.

These strains can be found, for example, in the American Type Culture Collection (ATCC; Addr
ess: PO Box 1549, Manassas, VA 20108, United States of America). Each strain has a corresponding registration number, which can be used to order (see atcc.org). The accession number for each strain is listed in the American Type Culture Collection catalog.

The bacteria described herein are modified to overexpress a gene encoding a protein with L-threonine 3-dehydrogenase activity and a gene encoding a protein with 2-amino-3-oxobutanoate coenzyme A ligase activity. ing.

The term "protein with L-threonine 3-dehydrogenase activity" may mean a protein that catalyzes the following reaction: L-threonine + NAD ⁺ ⇔ L-2-amino-3-oxobutanoate + NADH + 2H ⁺ (Enzyme Commission (EC) number 1.1.1.103; Boylan SA and Dekker EE, L-Threonine dehydrogenase. Purification and properties of the homogeneous enzyme
from E. coli K-12, J. Biol. Chem., 1981, 256(4):1809-1815). For example, a protein having L-threonine 3-dehydrogenase activity is a protein having the amino acid sequence shown in SEQ ID NO: 2 and its homologue, which is a protein having the amino acid sequence shown in SEQ ID NO: 2, and a homolog thereof, which has the NAD+ of L-threonine to L-2-amino-3-oxobutanoate.
It can mean something that catalyzes a dependent oxidation reaction. The activity of proteins with L-threonine 3-dehydrogenase activity can be determined by colorimetrically assessing the formation of aminoacetone from L-threonine or by measuring the formation of NADH using a spectrophotometer. (See Boylan SA and Dekker EE, 1981 and references therein).
The term "2-amino-3-oxobutanoate coenzyme A protein with ligase activity" refers to the following reaction: glycine + acetyl-coenzyme A ⇔ L-2-amino-3-oxobutanoate + coenzyme A +
Can refer to a protein that catalyzes H ⁺ (EC 2.3.1.29; acetyl-coenzyme A is also referred to as Ac-CoA; Mukherjee JJ and Dekker EE, Purification, properties, and
N-terminal amino acid sequence of homogeneous E. coli 2-amino-3-ketobutyrate CoA ligase, a pyridoxal phosphate-dependent enzyme, J. Biol. Chem., 1987, 262:14441-14447). For example, a protein having 2-amino-3-oxobutanoate coenzyme A ligase activity is a protein having the amino acid sequence shown in SEQ ID NO: 4 and its homolog, which is a protein that has the amino acid sequence shown in SEQ ID NO: 4 and its homologue, which is a protein that has the amino acid sequence shown in SEQ ID NO: 4 and its homologue. Can mean something that catalyzes a cleavage reaction. The activity of proteins with 2-amino-3-oxobutanoate coenzyme A ligase activity was determined by colorimetrically evaluating the formation of aminoacetone from glycine and Ac-CoA or by coenzyme A (also referred to as CoA). It can be determined by observing the condensation reaction of '-dithiobis-(2-nitrobenzoic acid) at 412 nm (see, for example, Mukherjee JJ and Dekker EE, 1987 and references therein).

Protein concentration can be determined by the Bradford protein assay or the Lowry method using Coomassie dye with bovine serum albumin (BSA) as a standard (Bradford M.M., Anal. Biochem., 1976, 72:248-254; Lowry O.H. et al. al., J. Biol. Chem., 1951, 193:265-275).

Examples of proteins having L-threonine 3-dehydrogenase activity include a protein having the amino acid sequence shown in SEQ ID NO:2. The amino acid sequence shown in SEQ ID NO: 2 can be encoded by the base sequence shown in SEQ ID NO: 1, which corresponds to the tdh gene. That is, examples of proteins having L-threonine 3-dehydrogenase activity include the tdh gene. The tdh gene of E. coli is L-threonine 3-dehydrogenase TDH, NAD(P)-binding (KEGG, Kyoto Encyclopedia of Genes and Genomes, entry No. b3616; Protein Knowledgebase, UniProtKB/Swiss-Prot, accession No. P07913 ). The tdh gene (GenBank, accession No. NC_000913.3; nucleotide positions: 3790320 to 3791345, complement; Gene ID: 948139) is located between the kbl gene and waaH gene in the chromosome of the E. coli K-12 strain. Located on the chain. The nucleotide sequence of the tdh gene (SEQ ID NO: 1) and the amino acid sequence of the TDH protein encoded by the E. coli tdh gene (SEQ ID NO: 2) are known. In addition, homologs of TDH from various bacterial species, such as E. coli (identity: 100%), Shigella flexneri (identity: 99%), Salmonella enteric (identity: 98%) with TDH of SEQ ID NO: 2 ), Klebsiella pneumonia
(identity: 97%), Enterobacter cloacae (identity: 96%), P. ananatis (identity: 86%); Enterobacteriaceae including species; Burkholderia mallei (identity: 77%) and Burkholderiaceae, including the species Paraburkholderia xenovorans (identity: 76%); Rhizobiaceae, including the species Rhizobium etli (identity: 71%); Xanthomonas axonopodis (identity: 64%) Homologues from bacteria belonging to the family Xanthomonadaceae, which includes species, are also known (e.g., the NCBI database,
National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/protein/). Therefore, examples of proteins having L-threonine 3-dehydrogenase activity include proteins that are homologs of the protein having the amino acid sequence shown in SEQ ID NO:2.

Examples of proteins having 2-amino-3-oxobutanoate coenzyme A ligase activity include a protein having the amino acid sequence shown in SEQ ID NO: 4. The amino acid sequence shown in SEQ ID NO: 4 can be encoded by the base sequence shown in SEQ ID NO: 3, which corresponds to the kbl gene. That is, the kbl gene is an example of a protein having 2-amino-3-oxobutanoate coenzyme A ligase activity. The E. coli kbl gene is 2-amino-3-oxobutanoate coenzyme A ligase KBL (also known as 2-amino-3-ketobutyrate coenzyme A ligase, 2-amino-3-oxobutanoate glycine-lyase (CoA-acetylating), glycine C-acetyltransferase, aminoacetone synthetase, aminoacetone synthase) (KEGG, entry No. b3617; Protein Knowledgebase, UniProtKB/Swiss-Prot, accession No. P0AB77). The kbl gene (GenBank, accession No. NC_000913.3; nucleotide positions: 3791355 to 3792551, complement; Gene ID: 948138) is located between the yibB gene and the tdh gene in the chromosome of the E. coli K-12 strain. Located on the chain. The nucleotide sequence of the kbl gene (SEQ ID NO: 3) and the amino acid sequence of the KBL protein encoded by the E. coli kbl gene (SEQ ID NO: 4) are known. Furthermore, homologs of KBL from various bacterial species, such as E. coli (identity: 100%), Shigella dysenteriae (identity: 99%), Citrobacter farmer (identity: 97%), have KBL of SEQ ID NO: 4. ), Salmonella enterica (identity: 97%), P. ananatis (identity: 82%); Enterobacteriaceae, which includes the species Serratia marcescens (identity: 88%); Yersiniaceae); Morganellaceae, including the species Xenorhabdus khoisanae (identity: 83%); Erwiniaceae, including the species Erwinia sp. 9145 (identity: 82%); Lysobacter spongiicola (identity: 67 Homologues from bacteria belonging to the Xanthomonadaceae family, including the species Stenotrophomonas maltophilia (identity: 66%); Therefore, examples of proteins having 2-amino-3-oxobutanoate coenzyme Aligase activity include proteins that are homologs of the protein having the amino acid sequence shown in SEQ ID NO: 4.

The term "bacteria has been modified to overexpress a gene encoding a protein with L-threonine 3-dehydrogenase activity and a gene encoding a protein with 2-amino-3-oxobutanoate coenzyme A ligase activity" In the engineered bacteria, the total activity of the corresponding gene product catalyzing the NAD+-dependent oxidation reaction of L-threonine to L-2-amino-3-oxobutanoate and 2-amino-3 compared to the unmodified strain. -oxobutanoate glycine and acetyl-coenzyme
or the expression level (i.e., the expression level) of the gene encoding a protein with L-threonine 3-dehydrogenase activity and the 2-amino -3-oxobutanoate coenzyme A ligase activity can mean that the bacterium has been modified in such a way that both the expression level (ie, the expression amount) of a gene encoding a protein with ligase activity is increased. The term "unmodified strain" may refer to a bacterial strain that may serve as a control for the above comparisons. The term "unmodified strain" is also referred to as "unmodified bacteria" or "unmodified bacterial strain." Non-modified strains include bacteria belonging to the genus Escherichia or Pantoea (E.
Examples include wild strains and parent strains of bacteria belonging to the Enterobacteriaceae family, including coli and P. ananatis). Specific examples of non-modified strains include E. coli strain MG1655 (ATCC 47076), W3110 (ATCC 27325), and P. ananatis strain AJ13355 (FERM BP-6614).

Bacteria that have been modified to overexpress a gene encoding a protein with L-threonine 3-dehydrogenase activity and a gene encoding a protein with 2-amino-3-oxobutanoate coenzyme A ligase activity are, for example, non-modified strains. Expression levels of genes encoding proteins with L-threonine 3-dehydrogenase activity and genes encoding proteins with 2-amino-3-oxobutanoate coenzyme A ligase activity compared to the wild-type strain or parent strain L-threonine to L-2-amino-3-oxobutanoate by increasing (i.e., enhancing) or by increasing the per molecule activity (also called specific activity) of the proteins encoded by those genes. Both the total activity of the corresponding gene product that catalyzes the NAD+-dependent oxidation reaction of Could be bacteria. An increase in the total activity of a protein can be measured, for example, as an increase in the activity of the protein per cell (which may be the average activity of the protein per cell). In bacteria, the activity of a protein having L-threonine 3-dehydrogenase activity per cell and/or the activity of a protein having 2-amino-3-oxobutanoate coenzyme A ligase activity per cell is lower than that of the same protein in an unmodified strain, for example. may be modified to increase the activity of 150% or more, 200% or more, or 300% or more.

The term "bacteria has been modified to overexpress a gene encoding a protein with L-threonine 3-dehydrogenase activity and a gene encoding a protein with 2-amino-3-oxobutanoate coenzyme A ligase activity" The expression level (i.e., the amount of expression) of a gene encoding a protein with L-threonine 3-dehydrogenase activity and 2-amino-3-oxobutanoate coenzyme compared to an unmodified strain (e.g., wild strain or parent strain) It may mean that the bacterium has been modified in such a way that both the expression level (ie, the expression amount) of a gene encoding a protein with A ligase activity is increased. Thus, the term "a gene is overexpressed" may be equivalent to the term "the expression of a gene is enhanced or increased" or the term "the expression level of a gene is enhanced or increased." An increase in the expression level of a gene can be measured, for example, as an increase in the expression level of the gene per cell (which may be the average expression level of the gene per cell). In bacteria, the expression level of a gene encoding a protein having L-threonine 3-dehydrogenase activity and/or the expression level of a gene encoding a protein having 2-amino-3-oxobutanoate coenzyme A ligase activity per cell is, for example, , may be modified to increase the expression level of the same gene by 150% or more, 200% or more, or 300% or more of the unmodified strain.

The above description regarding overexpression of genes encoding proteins with L-threonine 3-dehydrogenase activity and genes encoding proteins with 2-amino-3-oxobutanoate coenzyme A ligase activity applies to each of these genes independently. can be done.

A method of modifying a bacterium to overexpress both a gene encoding a protein having L-threonine 3-dehydrogenase activity and a gene encoding a protein having 2-amino-3-oxobutanoate coenzyme A ligase activity, and herein A method that can be used to enhance the expression of a gene encoding a protein having L-threonine 3-dehydrogenase activity and a gene encoding a protein having 2-amino-3-oxobutanoate coenzyme A ligase activity in the bacteria described in the book. may depend on the bacteria selected for modification. Any method of overexpressing a gene may be used as long as overexpression of the gene can be achieved. Therefore, a gene encoding a protein having L-threonine 3-dehydrogenase activity and 2-a
A gene encoding a protein with mino-3-oxobutanoate coenzyme A ligase activity may be overexpressed by one method of overexpressing the gene, or may be overexpressed by a variety of methods of overexpressing the gene.

Methods that can be used to enhance the expression of a gene include, but are not limited to, the number of copies of the gene, such as the number of copies of the gene in the bacterial chromosome and/or the autonomous replication carried by the bacterium. One example is increasing the copy number of a gene in a plasmid. The copy number of a gene can be increased, for example, by introducing the gene into the bacterial chromosome and/or by introducing an autonomously replicating plasmid containing the gene into the bacterium. Such an increase in gene copy number can be performed by genetic engineering techniques known to those skilled in the art.

Vectors include, but are not limited to, broad host range plasmids such as pMW118/119, pBR322, pUC19. Multiple copies of a gene encoding a protein having L-threonine 3-dehydrogenase activity and/or a gene encoding a protein having 2-amino-3-oxobutanoate coenzyme A ligase activity are generated by, for example, homologous recombination or Mu-driven integration. It can also be introduced into the chromosomal DNA of bacteria. Each gene is
Only one copy, two or more copies may be introduced. For example, multiple copies of a gene can be introduced into chromosomal DNA by performing homologous recombination targeting a sequence that exists in multiple copies in chromosomal DNA. Sequences that exist in multiple copies in chromosomal DNA include, but are not limited to, repetitive DNA and inverted repeats that exist at the ends of transposable elements. Furthermore, multiple copies of a gene can be introduced into chromosomal DNA by incorporating the gene into a transposon and transposing it. Mu-driven integration allows three or more copies of a gene to be introduced into chromosomal DNA in a single operation (Akhverdyan VZ et al., Biotechnol. (Russian), 2007, 3:3-20).

The bacterium described herein can be used after introduction of a gene encoding a protein with L-threonine 3-dehydrogenase activity and a gene encoding a protein with 2-amino-3-oxobutanoate coenzyme A ligase activity. These genes can be modified to overexpress them, as they are present in the cells. Bacteria can also be modified such that the activity of a protein having L-threonine 3-dehydrogenase activity and the activity of a protein having 2-amino-3-oxobutanoate coenzyme A ligase activity can be determined in the modified bacterium. In other words, any bacterium that does not naturally or naturally have a gene encoding a protein with L-threonine 3-dehydrogenase activity and a gene encoding a protein with 2-amino-3-oxobutanoate coenzyme A ligase activity, As described in the specification, the activity of a protein having L-threonine 3-dehydrogenase activity and the activity of a protein having 2-amino-3-oxobutanoate coenzyme A ligase activity can be determined in the modified bacteria, and A gene encoding a protein with L-threonine 3-dehydrogenase activity and a protein encoding a protein with 2-amino-3-oxobutanoate coenzyme A ligase activity, allowing bacteria to produce the tripeptide γ-Glu-Val-Gly. As long as the gene can be modified to overexpress it, it can be used.

The bacteria described herein have been modified to have a gene encoding a protein with L-threonine 3-dehydrogenase activity and a gene encoding a protein with 2-amino-3-oxobutanoate coenzyme A ligase activity. . A gene encoding a protein with L-threonine 3-dehydrogenase activity and a gene encoding a protein with 2-amino-3-oxobutanoate coenzyme A ligase activity are isolated from bacteria in such a way that the genes are present on different nucleic acid molecules. can be overexpressed in Alternatively, the genes can be introduced into bacteria such that the genes are on one nucleic acid molecule. For example, L-
The gene encoding a protein with threonine 3-dehydrogenase activity and the gene encoding a protein with 2-amino-3-oxobutanoate coenzyme A ligase activity may be present on one expression vector or chromosome. Alternatively, the genes may be present on two different expression vectors. Alternatively, one gene may be on one expression vector and the other gene on the chromosome.

Since bacteria belonging to the family Enterobacteriaceae are an example of the bacteria described herein, a gene encoding a protein having L-threonine 3-dehydrogenase activity and a 2-amino-3- A method for overexpressing a gene encoding a protein having oxobutanoate coenzyme A ligase activity will be described below.

A method for overexpressing a gene encoding a protein having L-threonine 3-dehydrogenase activity and a gene encoding a protein having 2-amino-3-oxobutanoate coenzyme A ligase activity in bacteria belonging to the family Enterobacteriaceae is This can be done by introducing a nucleic acid (DNA) having the following into Enterobacteriaceae bacteria. Methods for introducing nucleic acids (eg, genes, vectors, etc.) into Enterobacteriaceae bacteria include, but are not particularly limited to, genetic engineering techniques known to those skilled in the art. In the bacteria described herein, the gene encoding a protein having L-threonine 3-dehydrogenase activity and the gene encoding a protein having 2-amino-3-oxobutanoate coenzyme A ligase activity are extrachromosomal such as a plasmid. It can exist on a vector that replicates autonomously, or it can be integrated into a chromosome. Furthermore, as mentioned above, in order to construct the bacteria described herein, a gene encoding a protein having L-threonine 3-dehydrogenase activity and a 2-amino-3-oxobutanoate coenzyme
Introduction of a gene encoding a protein having A ligase activity and imparting or enhancing the ability to produce the tripeptide γ-Glu-Val-Gly can be carried out in any order.

Other methods that can be used to enhance the expression of genes encoding proteins with L-threonine 3-dehydrogenase activity and genes encoding proteins with 2-amino-3-oxobutanoate coenzyme A ligase activity include , increasing the expression level of those genes by modifying their expression control regions. The expression control regions of those genes can be modified, for example, by introducing native and/or modified foreign expression control regions into the native expression control regions of those genes. can. The term "expression control region" is also referred to as "expression control sequence." To enhance the expression of genes encoding proteins with L-threonine 3-dehydrogenase activity and/or 2-amino-3-oxobutanoate coenzyme A ligase activity when they form an operon structure. Methods that can be used to increase the expression level of an operon containing those genes include, for example, increasing the expression level of the operon by modifying the expression control region of the operon. ) can be carried out by replacing the expression control region of ) with a native and/or modified foreign expression control region. In this method, one or more genes in the operon, including a gene encoding a protein with L-threonine 3-dehydrogenase activity and a gene encoding a protein with 2-amino-3-oxobutanoate coenzyme A ligase activity, are expression can be simultaneously enhanced.

Expression control regions include promoters, enhancers, attenuators and termination signals, anti-termination signals, ribosome binding sites (RBS), and other expression control elements (
Examples include regions to which repressors or inducers bind, and/or binding sites for transcription and translation control proteins in transcribed mRNA, for example. Such control regions are described, for example, in Sambrook J., Fritsch EF and Maniatis T., "Molecular Cloning: A Laboratory Manual", ^2nd ed., Cold Spring Harbor Laboratory Press (1989). Modification of the expression control region of a gene may be combined with an increase in the copy number of the gene (e.g. Akhverdyan VZ et al., Appl. Microbiol. Biotechnol., 2011, 91:857-871; Tyo KEJ et al., Nature Biotechnol., 2009, 27:760-765).

Examples of promoters suitable for enhancing the expression of genes encoding proteins with L-threonine 3-dehydrogenase activity and genes encoding proteins with 2-amino-3-oxobutanoate coenzyme A ligase activity include their Examples include strong promoters that are stronger than the original promoter of a gene. For example, lac promoter, trp promoter, trc promoter, tac promoter, tet promoter, araBAD promoter, rpoH
The msrA promoter, the Pm1 promoter (from the genus Bifidobacterium), and the lambda phage _PR and _PL promoters are all known as strong promoters. Strong promoters can be used that give high levels of gene expression in bacteria belonging to the Enterobacteriaceae family. Alternatively, the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter region of a gene to obtain a stronger promoter function, thereby increasing the transcription level of the gene located downstream of the promoter. be able to. Additionally, substitutions of several nucleotides in the Shine-Dalgarno (SD) sequence and/or the spacer between the SD sequence and the start codon, and/or the sequence immediately upstream or downstream of the start codon in the ribosome binding site It is known that it greatly affects translation efficiency. For example, a 20-fold range of expression levels has been found depending on the nature of the three nucleotides preceding the start codon (Gold L. et al., Annu. Rev. Microbiol., 1981, 35:365 -403; Hui A. et al., EMBO J., 1984, 3:623-629).

The number of copies of a gene, or the presence or absence of a gene, can be determined by, for example, restriction treatment of chromosomal DNA followed by Southern blotting using probes based on the gene sequence, or fluorescence in situ hybridization (FISH). , can be measured.
The level of gene expression can be determined by measuring the amount of mRNA transcribed from the gene using a variety of well-known methods such as Northern blotting and quantitative RT-PCR. The amount of protein encoded by a gene can be measured by known methods such as SDS-PAGE followed by immunoblotting assay (Western blotting analysis) or mass spectrometry of a protein sample.

Methods for recombinant molecular manipulation and molecular cloning of DNA, such as preparation of plasmid DNA, cutting of DNA, ligation of DNA, transformation of DNA, selection of oligonucleotides as primers, introduction of mutations, etc., are within the skill of those skilled in the art. It may be a well-known conventional method. Such methods are described, for example, in Sambrook J., Fritsch EF and Maniatis T., “Molecular Cloning: A Laboratory
Manual”, ^2nd ed., Cold Spring Harbor Laboratory Press (1989), or Green MR and Sambrook JR, “Molecular Cloning: A Laboratory Manual”, ^4th ed., Cold Spring Harbor Laboratory Press (2012), Bernard R. Glick, Jack J. Pasternak and Cheryl L. Patten, “Molecular Biotechnology: principles and applications of recombinant DNA”, ^4th ed., Washington, DC, ASM Press (2009).

Any manipulation method using recombinant DNA can be used, including conventional methods such as transformation, transfection, infection, conjugation, mobilization, and the like. Transformation, transfection, infection, conjugation, or mobilization of bacteria with DNA encoding a protein can confer upon the bacteria the ability to synthesize the protein encoded by the DNA. Methods of transformation, transfection, infection, conjugation, and mobilization include any method. For example, for efficient DNA transformation and transfection, a method has been reported in which recipient cells are treated with calcium chloride to increase the permeability of E. coli K-12 to cellular DNA (Mandel M. and Higa A., Calcium-dependent
bacteriophage DNA infection, J. Mol. Biol., 1970, 53:159-162). Specialization and/or
Or generalized transformation methods have been described (Morse ML et al., Transductio
n in Escherichia coli K-12, Genetics, 1956, 41(1):142-156; Miller JH, Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor La. Press, 1972). Other methods for random and/or targeted integration of DNA into host microorganisms, e.g. "Mu-driven integration/amplification" (Akhverdyan et al., Appl. Microbiol. Biotechnol., 2011, 91:857 -871), "Red/ET-driven integration" or "λRed/ET-mediated integration" (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA 2000, 97(12):6640-45; Zhang Y ., et al., Nature Genet., 1998, 20:123-128) can be applied. Furthermore, for multiple insertions of desired genes, Mu-driven replicative transposition (Akhverdyan et al., Appl. Microbiol. Biotechnol., 2011, 91:857-871) and recA-dependent transposition leading to amplification of the desired genes are required. In addition to chemically inducible chromosome evolution based on sexual homologous recombination (Tyo KEJ et al., Nature Biotechnol., 2009, 27:760-765), translocation,
Methods that utilize various combinations of site-specific and/or homologous Red/ET-mediated recombination and/or P1-mediated generalized transduction (e.g., Minaeva N. et al., BMC Biotechnology, 2008, 8:63 ; Koma D. et al., Appl. Microbiol. Biotechnol., 2012, 93(2):815-829).

A method for overexpressing a gene encoding a protein having L-threonine 3-dehydrogenase activity and a gene encoding a protein having 2-amino-3-oxobutanoate coenzyme A ligase activity in a bacterial species other than those belonging to the Enterobacteriaceae family. For bacteria belonging to the Enterobacteriaceae family, the methods described herein can be applied mutatis mutandis or methods known to those skilled in the art can be used. Furthermore, the use of general methods suitable for overexpression of genes in bacteria belonging to the family Enterobacteriaceae is within the ordinary skill in the art. Furthermore, methods suitable for overexpressing genes in bacteria belonging to the Enterobacteriaceae family can be modified as appropriate and used to overexpress genes in other bacterial species, and vice versa. Thus, the methods of overexpressing genes described herein may be applied to virtually any of the bacteria described herein.

The following describes mutants of genes encoding proteins with L-threonine 3-dehydrogenase activity and mutants of proteins with L-threonine 3-dehydrogenase activity, specifically, those mutants unique to E. coli. do. The following description of such gene and protein variants refers to genes and encoded proteins that are unique to bacterial species other than E. coli and encode proteins that have L-threonine 3-dehydrogenase activity; It can be applied mutatis mutandis to any gene and protein, including genes encoding and encoded proteins for proteins having 2-amino-3-oxobutanoate coenzyme A ligase activity.

There can be differences in DNA sequences between bacterial families, genera, species, or strains. Therefore, genes encoding proteins having L-threonine 3-dehydrogenase activity are not limited to genes having the base sequence shown in SEQ ID NO: 1, but genes having the mutant base sequence of SEQ ID NO: 1 and L-threonine 3-dehydrogenase activity. - A gene encoding a protein having dehydrogenase activity may be included. Similarly, proteins having L-threonine 3-dehydrogenase activity are not limited to proteins having the amino acid sequence shown in SEQ ID NO: 2, but proteins having the mutant amino acid sequence of SEQ ID NO: 2 and having L-threonine 3-dehydrogenase activity. It may also include genes having the following. Examples of such mutant base sequences or mutant amino acid sequences include genes encoding proteins having L-threonine 3-dehydrogenase activity as exemplified above, homologues of proteins having L-threonine 3-dehydrogenase activity as exemplified above, and artificial genes. Variants include.

The term "variant protein" may refer to a protein having a variant amino acid sequence of SEQ ID NO:2.

Specifically, the term "variant protein" refers to a substitution, deletion, insertion, and/or addition of one or several amino acid residues compared to the amino acid sequence shown in SEQ ID NO: 2. In any case, a protein with one or more mutations in its sequence that retains the L-threonine 3-dehydrogenase activity described herein or whose three-dimensional structure is similar to that of an unmodified protein (e.g. , a protein having the amino acid sequence shown in SEQ ID NO: 2, etc.). The number of mutations in a mutant protein depends on the position of the amino acid residue in the three-dimensional structure of the protein or the type of amino acid residue. The number of changes in the variant protein is not strictly limited, but may range from 1 to 50 in SEQ ID NO:2, in another example 1 to 30, in another example 1 to 15, in another example 1 to 50. It may be 10, or in another example 1-5. This is because amino acids can have high homology with each other, and mutations between these amino acids either do not affect protein activity or do not significantly change the three-dimensional structure of the protein compared to the unmodified protein. It's possible because you don't. Therefore, the mutant protein is determined whether the L-threonine 3-dehydrogenase activity is maintained or the three-dimensional structure of the protein is significantly different from that of the unmodified protein (for example, a protein having the amino acid sequence shown in SEQ ID NO: 2). 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more of the entire amino acid sequence of SEQ ID NO: 2, unless changed to It may be a protein having an amino acid sequence with a homology of 98% or more, alternatively 99% or more, defined as the parameter "identity" when using the computer program BLAST.

Examples of substitutions, deletions, insertions, and/or additions of one or several amino acid residues include conservative mutations. Representative conservative variations may be conservative substitutions. Conservative substitutions include, but are not limited to, when the substitution site is an aromatic amino acid, Phe, Trp, Tyr, and when the substitution site is a hydrophobic amino acid, it is between Ala, Leu, Ile, between Val, between Glu, Asp, Gln, Asn, Ser, His, and Thr when the substitution site is a hydrophilic amino acid; between Gln and Asn when the substitution site is a polar amino acid; When the substitution site is a basic amino acid, it is between Lys, Arg, and His. When the substitution site is an acidic amino acid, it is between Asp and Glu. When the substitution site is an amino acid having a hydroxyl group, it is between Ser, This is a substitution that replaces each other between Thr. Examples of conservative substitutions are Ala to Ser or Thr, Arg to Gln, His or Lys, Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln. , Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Asn, Gln, Lys or Asp, Gly to Pro, Substitution of His with Asn, Lys, Gln, Arg or Tyr, Substitution of Ile with Leu, Met, Val or Phe, Substitution of Leu with Ile, Met, Val or Phe, Substitution of Lys with Asn, Glu, Gln, His or substitution to Arg, Met
to Ile, Leu, Val or Phe, Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala, Thr to Ser or Ala, Trp to Phe or Tyr , Tyr to His, Phe or Trp, and Val to Met, Ile or Leu.
Examples of substitutions, deletions, insertions, and/or additions of one or several amino acid residues also include non-conservative mutations, provided that the mutations occur at one or more different positions of the amino acid sequence. By the above second mutation, L-threonine 3-dehydrogenase activity is maintained or an unmodified protein (for example, a protein having the amino acid sequence shown in SEQ ID NO: 2,
etc.), the three-dimensional structure of the protein is compensated so that it does not change significantly.

Several computational methods can be used to assess the degree of protein or DNA homology, such as BLAST searches, FASTA searches, and the ClustalW method. BLAST (Basic Local
Alignment Search Tool, www.ncbi.nlm.nih.gov/BLAST/) search is a heuristic search algorithm used by the programs blastp, blastn, blastx, megablast, tblastn, and tblastx, which are “Methods for assessing the statistical significance of molecular sequence features”
by using general scoring schemes” Proc. Natl. Acad. Sci. USA, 1990, 87:2264-22
68; “Applications and statistics for multiple high-scoring segments in molecular sequences”. Proc. Natl. Acad. Sci. USA, 1993, 90:5873-5877) to attribute significance to findings. be. The computer program BLAST calculates three parameters: score, identity, and similarity. The FASTA search method is based on Pearson WR
(“Rapid and sensitive sequence comparison with FASTP and FASTA”, Methods Enzymol., 1990, 183:63-98). The ClustalW method was developed by Thompson JD et al.
(“CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice”, Nucleic Acids Res., 1994, 22:4673-4680). As used herein, the term "homology" may mean "identity" which is the identity between amino acid sequences or base sequences. Sequence identity between two sequences is calculated as the proportion of residues that match between the two sequences when the two sequences are aligned for maximum alignment. Note that "identity" between amino acid sequences specifically refers to the default Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional It may mean the sameness calculated using the score matrix adjustment). Furthermore, unless otherwise specified, "identity" between base sequences is calculated by blastn using the default Scoring Parameters (Match/Mismatch Scores = 1, -2; Gap Costs = Linear). It may mean the sameness.

The term “variant sequence” refers to any synonymous amino acid codon according to the Standard Genetic Code Book (see, e.g., Lewin B., “Genes VIII”, 2004, Pearson Education, Inc., Upper Saddle River, NJ 07458). can be used to mean a base sequence encoding a protein having L-threonine 3-dehydrogenase activity, such as a protein having the amino acid sequence shown in SEQ ID NO:2. Therefore, a gene encoding a protein having L-threonine 3-dehydrogenase activity may be a gene having a mutant base sequence due to the degeneracy of the genetic code.

In addition, the term "variant base sequence" refers to a base sequence complementary to the sequence shown in SEQ ID NO: 1, or a probe that can be prepared from the base sequence, as long as it encodes a protein having L-threonine 3-dehydrogenase activity. It can also mean a base sequence that can hybridize under stringent conditions. The term "stringent conditions" refers to specific hybrids, e.g. when using the computer program BLAST, the parameter "identity" is defined as homology of 60% or more, 65% or more, 70% or more, 75% 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more hybrids are formed, and non-specific hybrids, e.g. It may include conditions under which hybrids with low homology are not formed. Stringent conditions include, for example, 1× SSC (standard sodium citrate or standard sodium chloride) and 0.1% SDS (sodium dodecyl sulfate) at 60°C; Conditions include washing at 60°C or at 65°C with a salt concentration of 0.1×SSC, 0.1% SDS one or more times, and in other examples two or three times. Washing times may depend on the type of membrane used for blotting, but should generally be as recommended by the manufacturer. For example, the recommended washing time under stringent conditions for Amersham Hybond ^™ -N+ positively charged nylon membrane (GE Healthcare) is 15 minutes. The washing step can be carried out two or three times. As a probe, a portion of a sequence complementary to the sequence shown in SEQ ID NO: 1 may be used. Such probes are produced by PCR (polymerase chain reaction; White TJ et al., The polymerase chain reaction, Trends Genet., 1989, 5:185-189). The length of the probe is recommended to be greater than 50 bp, but can be chosen appropriately depending on the hybridization conditions, and is usually 100 bp.
~1 kbp. For example, if a DNA fragment with a length of approximately 300 bp is used as a probe, the post-hybridization wash conditions may be, for example,
×SSC, 0.1% SDS conditions can be used.

The term "variant base sequence" can also mean a base sequence encoding a mutant protein.

The base sequence of the E. coli-specific gene that encodes a protein with L-threonine 3-dehydrogenase activity has already been elucidated (see above). The unique gene or its variant base sequence is cloned from E. coli by PCR using E. coli DNA and oligonucleotide primers prepared based on the base sequence of the E. coli-specific tdh gene. Mutagenesis methods involve in vitro treatment of DNA containing the tdh gene, for example with hydroxylamine, or ultraviolet (UV) irradiation of E. coli containing the tdh gene or N-methyl-N'-nitro-nitrosoguanidine (NTG), which is commonly used for such treatments. ) or nitrous acid, or by chemical synthesis as a full-length gene construct. Genes specific to other organisms (such as Enterobacteriaceae bacteria other than E. coli) that encode proteins having L-threonine 3-dehydrogenase activity or their mutant base sequences can be similarly obtained.

The term "non-modified" when referring to genes (e.g., "non-modified genes") and proteins (e.g., "non-modified proteins"); ``natural'' or ``wild-type,'' which may be equivalent to the terms ``natural'' or ``wild-type,'' respectively, refers to an organism, specifically an unmodified strain of bacteria, e.g., Enterobacteriaceae. Naturally occurring in wild strains of bacteria (e.g., E. coli strain MG1655 (ATCC 47076), E. coli strain W3110 (ATCC 27325), P. ananatis strain AJ13355 (FERM BP-6614), etc.) It can refer to native genes and proteins that are expressed and/or naturally produced. An unmodified gene may encode an unmodified protein.

The term "native to" when referring to a protein or nucleic acid that is native to a particular biological species, such as a bacterial species, can mean a protein or nucleic acid that is native to that species. That is, a protein or nucleic acid unique to a particular species may refer to a protein or nucleic acid, respectively, that is naturally occurring in that species. Proteins or nucleic acids specific to a particular species can be isolated from that species and sequenced by methods known to those skilled in the art. Furthermore, since the amino acid or base sequence of a protein or nucleic acid isolated from the species in which the protein or nucleic acid exists, respectively, can be readily determined, the term "unique" when referring to a protein or nucleic acid Any means, such as genetic engineering techniques including recombinant DNA technology or It can also refer to proteins or nucleic acids obtained by chemical synthesis methods and the like. The term "protein" may include, but is not limited to, peptides, oligopeptides, polypeptides, proteins, enzymes, and the like. The term "nucleic acid" may include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and in particular, regulatory sequences, genes, intergenic sequences, including promoters, attenuators, terminators, etc. It may include sequences encoding sequences, signal peptides, pro-sites of proteins, artificial amino acid sequences, and the like. Specific examples of amino acid and base sequences and their homologs specific to various species are described herein. Specifically, as a protein specific to E. coli, TDH has the amino acid sequences shown in SEQ ID NOs: 2 and 4, respectively.
and KBL. Specific examples of E. coli-specific genes include the tdh and kbl genes, which have the base sequences shown in SEQ ID NOs: 1 and 3, respectively.

There are also other genes whose expression is altered either by downregulation of expression (i.e., attenuation of expression) or by overexpression of the gene, and such alterations may be caused by the addition of tripeptides during cultivation of the bacteria described herein. There may be a positive effect on the production of γ-Glu-Val-Gly. Bacteria may be modified to have such changes. The bacteria may specifically be modified to have any one or any combination of such modifications.

The term "a bacterium has been modified in such a way that the expression of a gene is attenuated" means that in the modified bacterium, the total activity of the corresponding gene product is reduced compared to the unmodified strain, or the expression of a gene is It can mean that the bacterium has been modified such that the level of expression (ie, the amount of expression) is reduced. In bacteria, the activity per cell of the protein encoded by the target gene is reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the activity of the same protein in the unmodified strain. It may be modified as follows. Bacteria are modified so that the expression level of the target gene per cell is reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the expression level of the same gene in the unmodified strain. It's okay to be.

The above description regarding overexpression of a gene encoding a protein having L-threonine 3-dehydrogenase activity and a gene encoding a protein having 2-amino-3-oxobutanoate coenzyme A ligase activity can be applied mutatis mutandis to other genes.

Specific methods of attenuating gene expression or overexpressing genes are well known to those skilled in the art. Attenuation of gene expression can be achieved, for example, by disrupting or deleting the gene. Overexpression of genes can be achieved in the same manner as, for example, overexpression of genes encoding proteins with L-threonine 3-dehydrogenase activity and genes encoding proteins with 2-amino-3-oxobutanoate coenzyme A ligase activity.

Such changes include attenuating the expression of genes encoding enzymes that prevent glycine degradation or increase glutamate biosynthesis. These changes may include attenuating expression of the gcvP gene and attenuating expression of the sucAB operon gene. The gcvP gene encodes a component of the glycine cleavage system, which prevents glycine degradation through glycine decarboxylation (Yishai et al., ACS Synth Biol. 2017;6(9):1722-1731) . The sucAB gene encodes two subunits of 2-ketoglutarate dehydrogenase (KGDH). KGDH activity can be reduced by weakening the expression of the sucAB gene (eg, reducing the activity of the protein encoded by the gene). For example, it has been reported that attenuated expression of the sucAB gene reduced KGDH activity by 60%, leading to a three-fold increase in glutamate accumulation (US 7,604,979 B2). Reducing KGDH activity is beneficial for γ-Glu-Val-Gly production due to its positive impact on glutamate synthesis.

Such changes also include overexpression of genes encoding proteins involved in the production of L-valine and proteins involved in the excretion of the tripeptide γ-Glu-Val-Gly. These changes may include overexpression of the ilvGMEDA operon gene (US Patent No. 5,998,178 A) or the tolC gene. The tolC gene encodes the outer membrane proteins of various tripartite efflux complexes (Benz R. et al., TolC of Escherichia coli functions as an outer membrane channel, Zentralbl. Bakteriol., 1993, 278 (2-3):187-196). The ilvGMEDA operon may include an ilvG gene with mutations that result in regeneration of the activity of acetohydroxylic acid synthase II (AHAS II) encoded by the ilvG gene (Russian Patent No. 2212447 C2; US Patent No. 9,896,704 B2).

Bacteria can have specific properties in addition to those described above, such as various auxotrophies, drug resistance, drug sensitivity, drug dependence, etc., without departing from the scope of the invention.

2. Method The method described herein for producing the tripeptide γ-Glu-Val-Gly using bacteria involves cultivating the bacteria in a culture medium (culturing) to produce γ-Glu-Val-Gly. The method includes a step of producing, excreting or secreting, and/or accumulating γ-Glu-Val-Gly in a culture medium or cells, or both, and a step of recovering γ-Glu-Val-Gly from the culture medium and/or cells. The method may optionally include the step of purifying the tripeptide γ-Glu-Val-Gly of interest from the culture medium and/or the bacterial cells. γ-Glu-Val-Gly can be produced in the form described above. γ-Glu-Val-Gly can be produced in particular in free form or as a salt thereof or as a mixture thereof. For example, salts such as sodium salts, potassium salts, ammonium salts, etc. can be produced by the method described above. This is possible because amino acids can react under fermentation conditions with each other or with neutralizing agents such as inorganic or organic acids or alkaline substances to form salts through typical acid-base neutralization reactions. , this is a chemical characteristic of amino acids that is clear to those skilled in the art.

Cultivation of the bacteria and recovery and optional purification of the tripeptide γ-Glu-Val-Gly from the medium etc. can be carried out in the same manner as conventional fermentation methods for producing L-amino acids using microorganisms. The culture medium may be a synthetic or natural medium, such as typical media containing carbon sources, nitrogen sources, sulfur sources, phosphorous sources, inorganic ions, and other organic and inorganic components as required. Carbon sources include sugars such as glucose, sucrose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose, starch hydrolysates, alcohols such as ethanol, glycerol, mannitol, and sorbitol, gluconic acid, and fumaric acid. , organic acids such as citric acid, malic acid, and succinic acid, fatty acids, and the like can be used. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen such as soybean hydrolyzate, ammonia gas, ammonia water, and the like can be used. Furthermore, peptone, yeast extract, meat extract, malt extract, corn steep liquor, etc. can also be used. The medium can contain one or more of these nitrogen sources. As a sulfur source, ammonium sulfate,
Examples include magnesium sulfate, iron sulfate, manganese sulfate, and the like. The medium may include a phosphorus source in addition to carbon, nitrogen, and sulfur sources. As the phosphorus source, phosphoric acid polymers such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, pyrophosphoric acid, etc. can be used. Vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12, as well as other necessary substances, such as adenine, nucleic acids such as RNA, amino acids, peptones, casamino acids, and organic nutrients such as yeast extract. , may be present in appropriate amounts (even trace amounts). In addition to these, small amounts of calcium phosphate, iron ions, manganese ions, etc. may be added if necessary.

Cultivation can be carried out under conditions suitable for culturing the bacteria selected in the method for producing the tripeptide γ-Glu-Val-Gly. For example, culturing can be carried out under aerobic conditions for 16-72 hours, or 32-48 hours. The culture temperature during culture can be controlled within the range of 30 to 45°C or 30 to 37°C. The pH can be adjusted between 5 and 8, or between 6 and 7.5. pH can be adjusted by using inorganic or organic acidic or alkaline substances, such as urea, calcium carbonate, or ammonia gas.

After culturing, the tripeptide γ-Glu-Val-Gly can be recovered from the culture medium. Specifically, the tripeptide γ-Glu-Val-Gly present outside the bacterial cells can be recovered from the culture medium. In addition, after culturing, the tripeptide γ-Glu-Val-Gly can be recovered from the bacterial cells. Specifically, the bacterial cells are crushed and the bacterial cells and the crushed bacterial cell suspension (also called cell debris) are collected. The tripeptide γ-Glu-Val-Gly can be recovered from the supernatant by removing the solid content such as the supernatant. The microbial cells can be disrupted by a well-known method such as ultrasonic disruption using high-frequency sound waves. Removal of solids can be carried out, for example, by centrifugation or membrane filtration. The tripeptide γ-Glu-Val-Gly can be recovered from the culture medium, supernatant, etc. by conventional methods such as concentration, crystallization, ion-exchange chromatography, medium-pressure or high-pressure liquid chromatography, or a combination thereof. It can be implemented by technology.

Hereinafter, the present invention will be explained more specifically using the following non-limiting examples.

Example 1: Construction of γ-EVG producing strain of E. coli
E. coli MG1655 P _L -ilvG*MEDA ΔicdC::P _tac4071 φ10-gshA50 attB φ80::KmR-P _tac4071φ10 -gshB strain ^M165F (L1029-1) was constructed as a model γ-EVG producing strain. For this purpose, the MG1655 ilvG*MEDA ilvH** strain was used as starting material. The MG1655 ilvG*MEDA ilvH** strain can be constructed from the E. coli K-12 strain substr. MG1655 (ATCC 47076) using conventional recombinant methods and/or genetic chemical synthesis methods. The MG1655 ilvG*MEDA ilvH** strain has a mutation in the ilvG gene that restores the frameshift of the wild-type ilvG gene (specifically, a two base pair (AA) insertion at position 981 from the start of the gene, upstream of the sequence TGActggca). The ilvG5 mutation has the wild-type protein sequence...PLNQ& is replaced with...PLNQNDW...), thereby restoring acetohydroxyacid synthase II (AHAS II) activity (Russian Patent No. 2212447 C2; US Patent No. 9,896,704 B2), two mutations resulting in the substitution of ¹⁷ Ser with Phe and the substitution of ¹⁴ Gly with Asp in the small subunit of acetolactate synthase III (AHAS III) were also conferred on the ilvH gene. (US Patent No. 6,737,255 B2). Datsenko and Wanner (Datsenko KA and
Shine-Dalgarno was modified using a method called "λRed-dependent integration" developed by Wanner BL, Proc. Natl. Acad. Sci.
The native ilvG*MEDA operon was replaced with an artificial control region containing the P _L promoter of phage λ linked to the sequence SD1 (SD sequence from the pET22 plasmid). Following this procedure, PCR primers P1 (SEQ ID NO: 5) and P2 (SEQ ID NO: 6) were constructed. Oligonucleotide P1 (SEQ ID NO: 5) is homologous to the upstream region of the ilvG gene and the flanking region of the chloramphenicol resistance gene (cat), which was obtained from the chromosomal DNA of BW25113 cat-P _L -yddG. There is. The acquisition of BW25113 cat-P _L -yddG is described in detail (EP1449918A1, Russian patent RU2222596 C1). The BW25113 cat-P _L -yddG strain was used as a template for PCR. Oligonucleotide P2 (SEQ ID NO: 6) has homology to both the ilvG region and the region downstream of the _PL promoter, which was obtained from the template chromosome and contains the SD1 sequence. PCR conditions were as follows: denaturation at 95°C for 3 min; profile for the first 2 cycles: 1 min at 95°C, 30 s at 34°C, 80 s at 72°C; profile for the last 30 cycles. : 95°C for 30 seconds, 50°C for 30 seconds, 72°C for 80 seconds; final step: 72°C for 5 minutes. The resulting DNA fragment (1957 bp; SEQ ID NO: 7) was purified on an agarose gel and used for electroporation into E. coli strain MG1655 containing the helper plasmid pKD46 carrying the temperature-sensitive replicon. Plasmid pKD46 (Datsenko, KA and Wanner, BL, Proc. Natl. Acad. Sci. USA, 2000, 97 (12):6640-45) is a 2,154 nt (31088 -33241) and contains the genes of the λRed homologous recombination system (gamma, beta, exo genes) under the control of the arabinose-inducible _ParaB promoter. Plasmid pKD46 is required to integrate the DNA fragment into the bacterial chromosome.

Electrocompetent cells were prepared as follows. E. coli MG1655 strain was grown in LB medium containing ampicillin (100 mg/L) (Sambrook, J. and Russell, DW, “Molecular Cloning: A Laboratory Manual”, 3rd ed., Cold Spring Harbor Laboratory Press (2001)). The culture was grown overnight at 30°C in 5 mL of SOB medium containing ampicillin (100 mg/L) and L-arabinose (1 mM) (Sambrook, J. Fritsch, EF and Maniatis, T.,
“Molecular Cloning: A Laboratory Manual”, 2nd ed., Cold Spring Harbor Laboratory Press (1989)) was diluted 100 times. Cells were incubated at 30℃ with aeration (250 rpm) to OD600.
The cells were grown to approximately 0.6, concentrated 100 times, and made electrocompetent by washing three times with ice-cold deionized water. Electroporation was performed using 200 μl of bacterial cells and approximately 100 ng of DNA fragment (SEQ ID NO: 7). The cells were then transferred to 1 mL of SOC medium (Sambrook, J. Fritsch, EF and Maniatis, T., “Molecular Cloning: A Laboratory
Manual”, 2nd ed., Cold Spring Harbor Laboratory Press (1989)) at 37°C for 2.5 hours, and plated on plates containing LB medium, agar (1.5%), and chloramphenicol (20 μg/mL). Chloramphenicol-resistant (Cm ^R ) recombinants were selected by inoculation and growth at 37°C. Then, to shed the pKD46 plasmid, they were grown at 42°C in L-agar containing Cm (20 μg/mL). After one passage, each resulting colony was tested for ampicillin sensitivity.The strain MG1655 cat-PL-SD1-ilvG*MEDA was thus obtained.

The native regulatory region of the ilvG*MEDA operon was replaced with the P _L-SD1 promoter labeled with the chloramphenicol resistance gene using locus-specific primers P3 (SEQ ID NO: 8) and P4 (SEQ ID NO: 9). This was confirmed by the PCR used. Conditions for PCR validation were as follows: denaturation at 95°C for 3 min; profile of 25 cycles: 30 s at 95°C, 30 s at 59°C, 1 min at 72°C; final step: at 72°C. 7 minutes. The DNA fragment obtained by the reaction using the bacterial cells of strain MG1655 as a template was 566 bp in length (SEQ ID NO: 10). The DNA fragment obtained by the reaction using the bacterial cells of the MG1655 cat-P _L-SD1 -ilvG*MEDA strain as a template was 2055 bp in length (SEQ ID NO: 11). In order to remove the CmR marker from the MG1655 cat-P _L-SD1 -ilvG*MEDA strain, the bacterial cells were transformed with plasmid pMW118-int-xis (Ap ^R ) (WO2005/010175). Ap ^R clone was transferred to LB-agar containing 150 mg/L ampicillin.
Grown on plates at 30°C. Several dozen Ap ^R clones were picked up and tested for chloramphenicol sensitivity. Plasmid pMW118-int-xis was removed from Cm ^S cells by culturing on LB agar plates at 42°C. In this way, the MG1655 P _L-SD1 -ilvG*MEDA strain was obtained.

γ-Glutamate-cysteine ligase (GshA) catalyzes the first step of γ-EVG biosynthesis. To increase the expression of the gshA gene encoding GshA, the expression cassette ΔicdC::KmR-P _tac4071φ10 -gshA50 (gshA50 means GshA ^L135F/Q144A (US2016326510 A1)) was transduced with P1 (Sambrook et al. MG1655 P _L-SD1 -ilvG*MEDA by “Molecular Cloning A Laboratory Manual, 2nd Edition”, Cold Spring Harbor Laboratory Press (1989)
introduced into the stock. Construction of the expression cassette ΔicdC::KmR-P _tac4071φ10 -gshA50 is described in Reference Example 3. Kanamycin-resistant transductants were selected and verified by PCR using locus primers P5 (SEQ ID NO: 12) and P6 (SEQ ID NO: 13). Conditions for PCR validation were as follows: 3 min denaturation step at 95°C; 25 cycle profile: 30 s at 95°C, 30 s at 55°C, 1 min at 72°C; final step: 72°C. 7 minutes. In the reaction using the bacterial cells of the parent strain MG1655 P _L-SD1 -ilvG*MEDA as a template, no DNA fragments were found. The length of the DNA fragment (SEQ ID NO: 14) obtained in a reaction using the bacterial cells of the MG1655 P _L-SD1 -ilvG*MEDA ΔicdC::P _tac4071φ10 -gshA50 strain as a template was 1850 nt. The kanamycin resistance (KmR) marker was removed from MG1655 P _L-SD1 -ilvG*MEDA ΔicdC::KmR-P _tac4071φ10 -gshA50 as described above. As a result, the MG1655 P _L-SD1 -ilvG*MEDA ΔicdC::P _tac4071φ10 -gshA50 strain was obtained.

Glutathione synthetase (GshB) catalyzes the second step of γ-EVG biosynthesis. To overexpress glutathione synthetase, the cassette attB φ80::KmR-P _tac4071φ10 -gshB ^M165F (Reference Example 4) encoding a mutant glutathione (γ-EVG) synthetase with improved selectivity for γ-EV substrates was added to P1. It was introduced into the MG1655 P _L-SD1 -ilvG*MEDA ΔicdC::P _tac4071φ10 -gshA50 strain by transduction. The donor strain MG1655 attB φ80::KmR-P _tac4071φ10 -gshB ^M165F can be constructed as detailed in Reference Example 1. KmR transduction bodies were selected and verified by PCR using locus primers P7 (SEQ ID NO: 33) and P8 (SEQ ID NO: 34). Conditions for PCR validation were as follows: 3 min denaturation step at 95°C; 25 cycle profile: 30 s at 95°C, 30 s at 61°C, 1 min at 72°C; final step: 72°C. 7 minutes. In the reaction using the bacterial cells of the parent strain MG1655 P _L-SD1 -ilvG*MEDA ΔicdC::P _tac4071φ10 -gshA50 as a template, no DNA fragments were found. MG1655 P _L-SD1 -ilvG*MEDA ΔicdC::P _tac4071φ10 -gshA50 attB phi80::KmR-Ptac4071φ10-gshB*Length of the DNA fragment (SEQ ID NO: 36) obtained in a reaction using the cells of the M165F strain as a template was 1917 nt. As a result, MG1655 P _L -ilvG*MEDA ΔicdC
::P _tac4071φ10 -gshA50 attB φ80::KmR-P _tac4071φ10 -gshB strain ^M165F (L1029-1) was obtained.

Example 2: Positive effects of kbl-tdh operon overexpression on γ-EVG production To provide γ-EVG precursors, the control region of the kbl-tdh operon was modified, i.e.
The native promoter region of the kbl-tdh operon was transformed into phage λ by the Red-dependent integration described above (Datsenko, KA and Wanner, BL, Proc. Natl. Acad. Sci. USA, 2000, 97 (12):6640-45). was replaced with the _PL promoter. Follow this procedure to PCR primer P9
(SEQ ID NO: 15) and P10 (SEQ ID NO: 16) were constructed. Oligonucleotide P9 (SEQ ID NO: 15) was used as a template for PCR in the region upstream of the kbl gene in the chromosomal DNA of the MG1655 cat-P _L - _SD1 -ilvG*MEDA strain (Example 1) and chloramphenicol resistance. There is homology in adjacent regions of the gene. Oligonucleotide P10 (SEQ ID NO: 16) has homology to both the kbl region in the chromosome of the MG1655 cat-P _L - _SD1 -ilvG*MEDA strain and the region downstream of the P _L promoter. PCR conditions were as follows: denaturation at 95°C for 3 min; profile for the first 2 cycles: 1 min at 95°C, 30 s at 34°C, 80 s at 72°C; profile for the last 30 cycles. : 30 seconds at 95℃, 30 seconds at 50℃, 80 seconds at 72℃; final step: 5 minutes at 72℃. The obtained DNA fragment (1969 bp; SEQ ID NO: 17) was purified using "Silica Bead DNA Gel Extraction Kit" (Thermo Scientific) and used for electroporation of E. coli strain MG1655 containing plasmid pKD46. After electroporation, chloramphenicol-resistant recombinants were selected. The native regulatory region of the kbl-tdh operon was replaced with the P _L-SD1 promoter labeled with the Cm resistance gene using PCR using locus-specific primers P11 (SEQ ID NO: 18) and P12 (SEQ ID NO: 19). Confirmed by. Conditions for PCR validation were as follows: denaturation at 95°C for 3 min; profile of 30 cycles: 30 s at 95°C, 30 s at 59°C, 1 min at 72°C; final step: at 72°C. 7 minutes. The DNA fragment obtained by the reaction using the bacterial cells of strain MG1655 as a template was 482 nt in length (SEQ ID NO: 20). The DNA fragment obtained by the reaction using the bacterial cells of the MG1655 cat-P _L -kbl-tdh strain as a template was 2273 nt in length (SEQ ID NO: 21). In this way, the MG1655 cat-P _L-SD1 -kbl-tdh strain was obtained.

Next, the expression cassette cat-P _L-SD1 -kbl-tdh was transferred to the γ-EVG producing strain MG1655 P _L-SD1 -ilvG*MEDA ΔicdC::P _tac4071φ10 -gshA50 attB φ80::KmR-P _tac4071φ10 - by P1 transduction. Introduced to gshB ^M165F (L1029-1). As a result, MG1655 P _L-SD1 -ilvG*MEDA ΔicdC::P _tac4071φ10 -gshA50 attBφ80::KmR-P _tac4071φ10 -gshB ^M165F cat-P _L-SD1 -kbl-tdh strain (L1031-1)
I got it. When strain L1031-1 was evaluated in comparison with the parent strain L1029-1, the positive effect of kbl-tdh operon overexpression on γ-EVG production was demonstrated (Table 1), i.e., the production of γ-EVG was increased by 3.5 times. As expected, the production of γ-EV, a by-product, decreased.

Example 3: Positive effects of gcvP gene disruption on γ-EVG production In order to prevent glycine degradation by glycine decarboxylase reaction, the above-mentioned Red-dependent integration encodes components of the glycine cleavage system. The gcvP gene was deleted. Following this procedure, PCR primers P13 (SEQ ID NO: 22) and P14 (SEQ ID NO: 23) homologous to both the gcvP gene in the template plasmid and the region flanking the gene conferring kanamycin resistance were constructed. Plasmid pMW118-(λattL-Km-λattR) (EP2100957 A1) was used as a template for the PCR reaction. PCR conditions were as follows: denaturation step at 95°C for 3 min; profile for the first 2 cycles: 1 min at 95°C, 30 s at 34°C, 80 s at 72°C; Profile: 30 seconds at 95°C, 30 seconds at 50°C, 80 seconds at 72°C; final step: 5 minutes at 72°C. The obtained DNA fragment (1614 bp; SEQ ID NO: 24) was purified using "Silica Bead DNA Gel Extraction Kit" (Thermo Scientific) and used for electroporation of E. coli strain MG1655 containing plasmid pKD46. Kanamycin-resistant recombinants were selected and the deletion of the gcvP gene marked with the KmR gene in the selected mutants was verified by PCR using locus-specific primers P15 (SEQ ID NO: 25) and P16 (SEQ ID NO: 26). did. Conditions for PCR validation were as follows: 3 min denaturation step at 95°C; 25 cycle profile: 30 s at 95°C, 30 s at 59°C, 2 min at 72°C; final step: 72°C. 7 minutes. The DNA fragment obtained in the reaction using the chromosomal DNA of the parental gcvP ⁺ strain MG1655 as a template was 3136 nt in length (SEQ ID NO: 27). The DNA fragment obtained in the reaction using the chromosomal DNA of mutant strain MG1655 ΔgcvP::KmR as a template was 1803 nt in length (SEQ ID NO: 28). As a result, the MG1655 ΔgcvP::KmR strain was obtained. MG1655ΔgcvP::KmR strain, MG1655 P _L-SD1 -ilvG*MEDA ΔicdC::P _tac4071φ10 -gshA50 attBφ80::P _tac4071φ10 -gshB ^M165F cat-P _L-SD1 -kbl-tdh strain (this is from L1031-1 strain (obtained by excision of the KmR marker) was used as a donor for the P1-mediated introduction of a deletion of the gcvP gene into the KmR marker. As a result, MG1655 P _L -ilvG*MEDA ΔicdC::P _tac4071φ10 -gshA50 attB φ80::P _tac4071φ10 -gshB ^{M165
The F} cat-P _L-SD1 -kbl-tdhΔgcvP::KmR strain (L1033-1) was selected. Evaluation of the L1033-1 strain revealed the positive effect of gcvP gene disruption on γ-EVG production, and as expected, the by-product γ-EV was reduced (Table 2).

Example 4: Positive effect of ilvGMEDA operon expression on γ-EVG production
To demonstrate the effect of ilvGMEDA operon expression on γ-EVG production, MG1655P_L-ilvG*MEDA ΔicdC::P_tac4071φ10-gshA50 attB φ80::P_tac4071φ10-gshB^M165Fcat-P_L-SD1-kbl-tdh Expression cassette P of ΔgcvP::Km strain (L1033-1)_L-SD1-ilvG*MEDA to wild type ilvGMEDA oper
Replaced with For this, first, MG1655 P_L-ilvG*MEDA ΔicdC::P_tac4071φ10-gshA50 attB φ80::P_tac4071φ10-gshB^M165F P_L-SD1-kbl-tdhΔgcvP::KmR strain (L1034-1), Cm^R
Obtained from L1033-1 strain by marker deletion. Then, expression cassette P of strain L1034-1_L-SD1-ilvG*MEDA expression cassette P by P1 transduction_L-SD1Replaced with -ilvG*M-ΔilvE::cat-DA. MG1655 P used as donor strain_L-SD1-ilvG*M-ΔilvE::cat-DA strain is detailed in Reference Example 2
It can be constructed as described in . Thus, L1034-1 P, which requires isoleucine and valine for growth in minimal medium,_L-SD1-ilvG*M-ΔilvE::cat-DA strain was obtained.

Then L1034-1 P_L-SD1-ilvG*M-ΔilvE::Cat-DA strain chromosome expression cassette P_L-SD1-ilvG*M-ΔilvE::cat-DA was transformed into wild-type ilvG by P1 transduction from E. coli strain MG1655.
MEDA operon was substituted, and prototrophic transduction bodies were selected on M9 minimal medium. MG1655 ΔicdC::P_tac4071φ10-gshA50 attB φ80::P_tac4071φ10-gshB^M165FP_L-SD1-kbl-tdh
Recovery of the wild-type ilvGMEDA operon in the ΔgcvP::KmR strain (L1040-1) was verified by PCR using locus-specific primers P3 (SEQ ID NO: 8) and P4 (SEQ ID NO: 9). Conditions for PCR validation were as follows: 5 min denaturation step at 94°C; 25 cycles of profile
Illumination: 30 seconds at 94°C, 30 seconds at 59°C, 1 minute at 72°C; final step: 7 minutes at 72°C. The DNA fragment obtained by the reaction using the bacterial cells of the parent strain as a template was 458 nt in length (SEQ ID NO: 29). The DNA fragment obtained by the reaction using the cells of strain L1040-1 as a template was 566 nt in length (SEQ ID NO: 10). Table 3?
As can be seen, efficient P in the chromosome of L1040-1 strain_L-SD1-By replacing the ilvGMEDA expression cassette with the wild-type ilvGMEDA operon, γ-EVG production is significantly reduced compared to the L1034-1 strain.
I put it down.

Example 5: Positive effect of sucAB expression on γ-EVG production
Reducing 2-ketoglutarate dehydrogenase (KGDH) activity was thought to be beneficial for γ-EVG production due to its positive impact on Glu synthesis. Downregulation of the sucAB gene, which encodes the two subunits of KGDH, alters the native promoter from P_tacAn artificial promoter derived from P_tac21(US 7,604,979 B2)
. In E. coli, the expression cassette cassette cat-P_tac21-sucAB has been demonstrated to reduce KGDH activity by 60%, thereby increasing Glu accumulation by 3-fold (US 7,604,979 B2). This cassette was transferred to γ-EVG producing strain MG1655P by P1 transduction._L-ilvG*MEDA ΔicdC::P_tac4071φ10-gshA50 attB φ80::P_tac4071φ10-gshB^M165FP_L-SD1-kbl-tdh It was also introduced into ΔgcvP::KmR (L1034-1). However, in fed-batch EVG cultures, no positive effect on γ-EVG or γ-EV accumulation was observed. However, when flasks are used for culture, downregulation of the sucAB gene
The positive effect of regulation (Table 4), that is, the effect of increasing γ-EVG production by approximately 2.5 times.
fruit was observed. Apparently, the difference in aeration conditions between the flask and jar was the main reason for the difference in the evaluation results.

Experimental procedure: Stock tube of bacterial cells (stored at -70 ^o C with 25% glycerol, 0.9% NaCl) L-Agar (yeast extract (Dia-M) 5 g/L, peptone (Dia-M) 10 g/L , NaCl 5 g/L, agar 15 g/L). Approximately half of the bacterial cells on the plate surface were inoculated into 40 mL of MS(+pyr) medium (Table 5), and cultured at 30°C for 24 hours on a rotary shaker. Additional glucose was then added to a final concentration of 20 g/L. After this addition, each strain was cultured at 30°C for 24 hours on a rotary shaker. Total culture time was 48 hours.

Example 6: Positive effects of tolC overexpression on γ-EVG production
To improve the efflux of γ-EVG, we modified the tolC gene (Benz R. et al., 1993), which encodes an outer membrane protein involved in various tripartite efflux complexes.
increased expression. For this, the expression cassette cat-P_Ltac-tolC (Reference Example 5) was transferred to γ-EVG producing strain MG1655 P by P1 transduction._L-SD1-ilvG*MEDA ΔicdC::P_tac4071φ10-gshA50 attB φ80::P_tac4071φ10-gshB^M165FP_L-SD1-kbl-tdh introduced into ΔgcvP::KmR (L1034-1)
Ta. Obtained strain L1034-1 cat-P_Ltac-tolC (L1036-1) was evaluated in comparison with the parent strain.
A positive effect of tolC overexpression on γ-EVG production was demonstrated, ie, more than 25% increase in γ-EVG production upon tolC overexpression (Table 6).

Reference example 1: Construction of cassette attB phi80::KmR-Ptac4071φ10-gshB ^M165F
The DNA fragment containing the gshB ^M165F gene was transferred from the plasmid pUC19-EcGshB*M165F (Reference Example 4) to the integration vector pAH162-λattL-TcR-λattR (Minaeva NI, et al. Dual-In/Out strategy for genes integration into bacterial chromosome : a novel approach to step-by-step construction of plasmid-less marker-less recombinant E. coli strains with predesigned genome structure. BMC Biotechnology, 2008, 8:63) using PstI/SacI restriction sites. Delivery plasmid pAH162-GshB*M165F was obtained. The gshB ^M165F gene was inserted into the native φ80 locus by φ80 integrase-mediated recombination (Minaeva NI, et al., 2008). Next, in order to obtain a high expression level, the regulatory region P _tac4071φ10 was introduced upstream of the gshB ^M165F gene by the Red-dependent integration method described above. Following this procedure, PCR primers P17 (SEQ ID NO: 30) and P18 (SEQ ID NO: 31) were constructed. Oligonucleotide P17 (SEQ ID NO: 30) is the gshB ^M165F in the chromosomal DNA of the MG1655 P _L -SD1-kbl-tdh ΔgcvP P _L -SD1-ilvG*MEDA ΔicdC::KmR-Ptac4071φ10-gshA50 strain, which was used as a PCR template. There is homology in the upstream region of the gene and adjacent regions of the KmR gene. Oligonucleotide P18 (SEQ ID NO: 31) is the gshB ^M165F region and P _tac4071φ10 control in the chromosome of the MG1655 P _L -SD1-kbl-tdh ΔgcvP P _L -SD1-ilvG*MEDA ΔicdC::KmR-P _tac4071 φ10-gshA50 strain. There is homology in both regions downstream of the region. PCR conditions were as follows: denaturation at 95°C for 3 min;
2-cycle profile: 95°C for 1 min, 34°C for 30 s, 72°C for 80 s; final 30-cycle profile: 95°C for 30 s, 50°C for 30 s, 72°C for 80 s; final step : 5 minutes at 72℃. The resulting 1721 bp DNA fragment (SEQ ID NO: 32) was purified using the “Silica Bead DNA Gel Extraction Kit”.
(Thermo Scientific) and used for electroporation of MG1655 attB phi80::gshB ^M165F strain containing plasmid pKD46. Select kanamycin-resistant recombinants and KmR
Introduction of the Ptac4071φ10 promoter labeled with the resistance gene was confirmed by PCR using locus-specific primers P7 (SEQ ID NO: 33) and P8 (SEQ ID NO: 34). Conditions for PCR validation were as follows: denaturation at 94°C for 5 min; profile of 25 cycles: 30 s at 94°C, 30 s at 59°C, 1 min at 72°C; final step: at 72°C. 7 minutes. The DNA fragment obtained by the reaction using the MG1655 strain as a template was 274 nt in length (SEQ ID NO: 35). The DNA fragment obtained in the reaction using the MG1655 attB phi80::KmR-Ptac4071φ10-gshB*M165F strain as a template was 1917 nt in length (SEQ ID NO: 36). As a result, the MG1655 attB phi80::KmR-Ptac4071φ10-gshB ^M165F strain was obtained.

Reference example 2: Construction of cassette P _L-SD1 -ilvG*M-ΔilvE::cat-DA Cassette P _L-SD1 -ilvG*M-ΔilvE::cat-DA was constructed by the Red-dependent integration method described above. . Following this procedure, PCR primers P19 (SEQ ID NO: 37) and P20 (SEQ ID NO: 38) were constructed. These primers have homology to both the flanking region of the ilvE gene and the gene conferring chloramphenicol resistance contained in the template plasmid pMW-attL-Cm-attR (PCT application WO 05/010175). PCR conditions were as follows: denaturation step at 95°C for 3 min; profile for the first 2 cycles: 1 min at 95°C, 30 s at 34°C, 80 s at 72°C; Profile: 30 seconds at 95°C, 30 seconds at 50°C, 80 seconds at 72°C; final step: 7 minutes at 72°C. The obtained DNA fragment (1713 bp; SEQ ID NO: 39) was purified with “Silica Bead DNA Gel Extraction Kit” (Thermo Scientific) and used for electroporation of MG1655 P _L-SD1 -ilvG*MEDA strain containing plasmid pKD46. did. Chloramphenicol-resistant recombinants were selected and the deletion of the Cm ^R gene-tagged ilvE gene in the selected mutants was performed using locus-specific primers P21 (SEQ ID NO: 40) and P22 (SEQ ID NO: 41). It was verified by PCR. Conditions for PCR validation were as follows: 5 min denaturation step at 94°C; 25 cycle profile: 30 s at 94°C, 30 s at 57°C, 1 min at 72°C; final step: 72°C. 7 minutes. The DNA fragment obtained in the reaction using parent strain MG1655 P _L-SD1 -ilvG*MEDA as a template was 1354 nt in length (SEQ ID NO: 42). The DNA fragment obtained by the reaction using the MG1655 P _L-SD1 -ilvG*M-ΔilvE::cat-DA strain as a template had a length of 2015 nt (SEQ ID NO: 43).

Reference Example 3: Construction of expression cassette ΔicdC::KmR-P _tac4071φ10 -gshA50
PCR was performed using pSF12-gshA*50 (described in US2016326510 A1) as a template and primer pair P23 (SEQ ID NO: 44) and P24 (SEQ ID NO: 45), and the chemically synthesized Ptac4071f10 fragment (
Scaffold nucleotides were added to the ends of the gshA*50 fragment for fusion PCR with SEQ ID NO: 46). The scaffolded gshA*50 fragment and the Ptac4071f10 fragment were then mixed and used as a template for fusion PCR using the primer pair P24 (SEQ ID NO: 45) and P25 (SEQ ID NO: 47) to construct the Ptac4071f10-gshA*50 fragment. . The Ptac4071f10-gshA*50 fragment was cloned into the XbaI restriction site of pMW118-attL-kan-attR plasmid (JP 2005-058227, WO2005/010175) by in-fusion technology (In-Fusion HD cloning Kit, Clontech). . The constructed plasmid was named pMW118-attL-kan-attR-Ptac4071f10-gshA*50. The KmR-Ptac4071f10-gshA50 cassette was amplified by PCR using pMW118-attL-kan-attR-Ptac4071f10-gshA*50 as a template and primer pair P26 (SEQ ID NO: 48) and P27 (SEQ ID NO: 49). The obtained DNA fragments were subjected to λ-red technology (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA 2000, 97(12):6640-45; Zhang Y., et al., Nature Genet., 1998 , 20:123-128) into E. coli K-12 MG1655 strain (ATCC 47076). The constructed strain was named MG1655ΔicdC::attL-kan-attR-Ptac4071f10-gshA*50 (carrying the cassette ΔicdC::KmR-P _tac4071φ10 -gshA50).

Reference Example 4: Construction of cassette attB φ80::CatR-P _tac4071φ10 -gshB ^M165F First, gshB expression plasmid pUC19-Plac-gshB was prepared. Using the chromosomal DNA of strain MG1655 as a template, the gshB fragment was amplified using primer pair P28 (SEQ ID NO: 50) and P29 (SEQ ID NO: 51). The gshB fragment was cloned into the XbaI site of pUC19 (New England Biolabs) by in-fusion technology (In-Fusion HD cloning Kit, Clontech). Next, PCR was performed using pUC19-Plac-gshB as a template and primer pair P30 (SEQ ID NO: 52) and P31 (SEQ ID NO: 53) to prepare pUC19-Plac-gshB*M165F. After digesting the obtained PCR fragment with DpnI to degrade the PCR template pUC19-Plac-gshB, the PCR solution was used to transform competent cells JM109 (available from Takara Bio) to transform the gshB*M165F expression plasmid pUC19- Plac-gshB*M165F was obtained.

PCR was performed using pUC19-Plac-gshB*M165F as a template and primer pair P32 (SEQ ID NO: 54) and P33 (SEQ ID NO: 55) for fusion PCR with the chemically synthesized Ptac4071f10 fragment (SEQ ID NO: 46). Scaffold nucleotides were added to the ends of the gshB*M165F fragment. The scaffolded gshB*M165F fragment and the Ptac4071f10 fragment were then mixed and used as a template for fusion PCR using the primer pair P25 (SEQ ID NO: 47) and P33 (SEQ ID NO: 55) to construct the Ptac4071f10-gshB*M165F fragment. . The Ptac4071f10-gshB*M165F fragment was cloned into the XbaI site of the pMW118-attL-cat-attR plasmid by in-fusion technology (In-Fusion HD cloning Kit, Clontech). The constructed plasmid was named pMW118-attL-cat-attR-Ptac4071f10-gshB*M165F. Primer pair P34 (SEQ ID NO: 56) using pMW118-attL-cat-attR-Ptac4071f10-gshB*M165F as a template.
and P35 (SEQ ID NO: 57) to amplify the CatR-Ptac4071f10-gshB*M165F cassette. The obtained DNA fragment was introduced into the MG1655ΔicdC::attL-kan-attR-Ptac4071f10-gshA*50 strain (Reference Example 3) using the λ-red technology. The constructed strain is MG1655ΔicdC::attL-kan-attR-Ptac4071f10-gshA*50 ΔgshB::attL-cat-attR-Ptac4071f10-gshB*M165F (holding cassette attB φ80::CatR-P _tac4071φ10 -gshB ^M165F ) It was named.

Reference example 5: Construction of expression cassette cat-P _Ltac -tolC
The tolC gene was overexpressed by the Red-dependent integration method (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-45). Following this procedure, PCR primers P36 (SEQ ID NO: 61) and P37 (SEQ ID NO: 62) have homology to both the flanking regions of the tolC gene and the flanking regions of the chloramphenicol resistance gene (CmR) and PLtac promoters in the template chromosome. ) was constructed. The chromosome of the MG1655 cat-P _L-tac -xylE strain (containing the hybrid P _Ltac promoter) was used as a template for the PCR reaction. The MG1655 cat-P _Ltac -xylE strain can be constructed as detailed in WO2006/043730. PCR conditions were as follows: denaturation at 95°C for 3 min; profile for the first two cycles: 1 at 95°C.
min, 30 s at 34°C, 80 s at 72°C; profile for last 30 cycles: 30 s at 95°C, 30 s at 50°C, 80 s at 72°C; final step: 5 min at 72°C. Obtained DNA fragment (1791 bp; SEQ ID NO: 58)
was purified using the "Silica Bead DNA Gel Extraction Kit" (Thermo Scientific) and used for electroporation of E. coli MG1655 strain containing plasmid pKD46. After electroporation, chloramphenicol-resistant recombinants were selected. PCR using locus-specific primers P38 (SEQ ID NO: 63) and P39 (SEQ ID NO: 64) revealed that the native control region of the tolC gene was replaced with the P _Ltac promoter region labeled with the Cm resistance gene. Confirmed by. The conditions for PCR validation were as follows: denaturation at 95°C for 3 min; profile of 30 cycles: 95°C for 30 s, 57°C for 30 s, 72°C for 1 min; final step: at 72°C. 7 minutes. The DNA fragment (SEQ ID NO: 59) obtained in a reaction using the bacterial cells of MG1655 strain as a template was 500 nt in length. The DNA fragment (SEQ ID NO: 60) obtained in a reaction using the bacterial cells of the MG1655 cat-P _Ltac -tolC strain as a template was 2279 nt in length. Plasmid pKD46 was then removed by culturing at 42°C. In this way, E. coli MG1655 strain carrying the expression cassette cat-P _Ltac -tolC was constructed.

Reference Example 6: EVG Culture In each of the above Examples, the following culture conditions were used for EVG culture.

All E. coli strains were maintained at 4°C on LB agar plates. LB agar (2%) was used for preincubation of all test strains overnight (17 h) at 37°C.

Seed culture was carried out for 5 hours in LB liquid medium (medium volume 50 mL) in a 750 mL Erlenmeyer flask in a GALLENCAMP shaker. The culture temperature was 37°C and the rotation speed was 240 rpm. One loop of biomass from the pre-cultured plate was used to inoculate the seed culture.

The main culture was performed in a 1 L S jar (ABLE Biot). The culture conditions were as follows:
Stirring was fixed at 1200 rpm, aeration was 1/1 vvm, temperature was 37°C, and pH 6.6 was maintained with NH ₃ gas. Dissolved oxygen levels were controlled as follows: If the DO level reached 0% (DO limiting condition occurred), after stopping the DO limitation, the agitation was reduced to maintain the DO level at 2%. did. If the DO level did not reach 0%, DO control was not performed. The medium volume was 300 mL, and the inoculum amount of the seed culture was 10%.

Table 7 shows the composition of the medium used for main culture.

After 6 hours of cultivation, glucose feed (concentration 700 g/L) was continuously added so that the glucose level in the culture was maintained within the range of 10-25 g/L. The total cultivation time of the main process is 24
It was time.

Reference Example 7: HPLC Analysis Similarly, in each of the above Examples, HPLC analysis was performed as follows. Analysis of γ-EVG was performed using high performance liquid chromatography (HPLC). A modified AccQ*Tag method with pre-column derivatization was used to analyze the samples.
Instrument: Agilent 1100 Column with fluorescence detector: Waters AccQ*Tag 3.9x150 mm (Part No. WAT052885)
Derivatization reagent: AccQ-Fluor Reagent Kit (WAT052880)
Derivatization conditions: 25 μL AccQ-Fluor Reagent Kit buffer, 5 μL sample, 20 μL
AccQ-Fluor Reagent Kit reagents; mix and incubate at 55°C for 10 minutes. Eluent:
A: 10% AccQ*Tag Eluent A (WAT052890) Aqueous solution B: 80% Acetonitrile (HPLC grade) Aqueous solution injection time: 38 minutes Injection volume: 5 μL
Detection: λ _ex - 250 nm, λ _em - 395 nm
Column temperature: 30°C
Flow rate: 0.8 mL/min
Gradient: Table 8

Although the invention has been described in detail with reference to illustrative embodiments thereof, it will be apparent to those skilled in the art that various modifications and equivalents can be made thereto without departing from the scope of the invention. Each of the documents mentioned above is incorporated herein by reference in its entirety.

The method of the invention is useful for producing the tripeptide γ-Glu-Val-Gly by bacterial fermentation.

Claims (10)

A method for producing γ-Glu-Val-Gly, comprising:
(i) Cultivate γ-Glu-Val-Gly-producing bacteria belonging to the Enterobacteriaceae family in a culture medium to inject γ-Glu-Val-Gly into the culture medium, the bacterial cells, or both. and (ii) recovering γ-Glu-Val-Gly from the culture medium or the bacterial cells, or both;
the bacterium is modified to overexpress a gene encoding a protein having L-threonine 3-dehydrogenase activity and a gene encoding a protein having 2-amino-3-oxobutanoic acid coenzyme A ligase activity;
The protein having L-threonine 3-dehydrogenase activity is selected from the group consisting of:
(A) Protein comprising the amino acid sequence shown in SEQ ID NO: 2;
(B) a protein containing an amino acid sequence containing substitutions, deletions, insertions, and/or additions of 1 to 30 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, and having L-threonine 3-dehydrogenase activity; and (C) a protein comprising an amino acid sequence having 90% or more identity to the entire amino acid sequence shown in SEQ ID NO: 2 and having L-threonine 3-dehydrogenase activity;
and,
The method, wherein the protein having 2-amino-3-oxobutanoic acid coenzyme A ligase activity is selected from the group consisting of:
(D) A protein containing the amino acid sequence shown in SEQ ID NO: 4;
(E) 2-amino-3-oxobutanoic acid coenzyme A ligase containing an amino acid sequence containing substitution, deletion, insertion, and/or addition of 1 to 30 amino acid residues in the amino acid sequence shown in SEQ ID NO: 4; and (F) a protein comprising an amino acid sequence having 90% or more identity to the entire amino acid sequence shown in SEQ ID NO: 4 and having 2-amino-3-oxobutanoic acid coenzyme A ligase activity. The gene encoding the protein having L-threonine 3-dehydrogenase activity is the tdh gene, and the gene encoding the protein having 2-amino-3-oxobutanoic acid coenzyme A ligase activity is the kbl gene. The method described in Section 1. The method according to claim 1 or 2,
The protein having L-threonine 3-dehydrogenase activity is encoded by a DNA selected from the group consisting of:
(a) DNA containing the base sequence shown in SEQ ID NO: 1;
(b) DNA comprising a base sequence that is complementary to the sequence shown in SEQ ID NO: 1 and a base sequence that can hybridize under stringent conditions,
The above stringent conditions are such that hybrids with 90% or more identity are formed and
is less than 90%, a condition under which no hybrid is formed ;
(c) DNA encoding a protein comprising an amino acid sequence containing substitutions, deletions, insertions, and/or additions of 1 to 30 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, wherein the protein is DNA that has L-threonine 3-dehydrogenase activity; and (d) DNA that is a variant base sequence of SEQ ID NO: 1 due to degeneracy of the genetic code;
and,
A method, wherein the protein having 2-amino-3-oxobutanoic acid coenzyme A ligase activity is encoded by a DNA selected from the group consisting of:
(e) DNA containing the base sequence shown in SEQ ID NO: 3;
(f) DNA comprising a base sequence that is complementary to the sequence shown in SEQ ID NO: 3 and a base sequence that can hybridize under stringent conditions,
The above stringent conditions are such that hybrids with 90% or more identity are formed and
is less than 90%, a condition under which no hybrid is formed ;
(g) DNA encoding a protein comprising an amino acid sequence containing substitutions, deletions, insertions, and/or additions of 1 to 30 amino acid residues in the amino acid sequence shown in SEQ ID NO: 4, wherein the protein is DNA having 2-amino-3-oxobutanoic acid coenzyme A ligase activity; and (h) DNA having a mutant base sequence of SEQ ID NO: 3 due to degeneracy of the genetic code. The gene encoding the protein having L-threonine 3-dehydrogenase activity and the gene encoding the protein having 2-amino-3-oxobutanoic acid coenzyme A ligase activity are each selected from the group consisting of: The method according to any one of claims 1 to 3, wherein the genes are overexpressed and the expression of these genes is increased compared to unmodified bacteria:
(i) increasing the copy number of that gene or those genes in said bacterium;
(ii) modifying the gene or the expression control region of those genes in the bacterium; and (iii) a combination thereof. The method according to any one of claims 1 to 4, wherein the bacterium is further modified by a method selected from the group consisting of:
(iv) weakening the expression of the gcvP gene;
(v) weakening the expression of the sucAB operon gene;
(vi) overexpressing the tolC gene; or (vii) overexpressing the ilvGMEDA operon gene; and (viii) a combination thereof. 6. The method according to claim 5, wherein the ilvGMEDA operon gene includes the ilvG gene encoding acetolactate synthase II, and the activity of the acetolactate synthase II is restored. The method according to any one of claims 1 to 6, wherein the bacterium belongs to the genus Escherichia or the genus Pantoea. 8. The method of claim 7, wherein the bacterium is Escherichia coli or Pantoea ananatis. According to any one of claims 1 to 8, the bacterium has been modified to overexpress a gene encoding γ-Glutamate-cysteine ligase (GshA) and a gene encoding Glutathione synthetase (GshB). the method of. 10. The method of claim 9, wherein the γ-Glutamate-cysteine ligase is selected from the group consisting of:
(A) Protein containing the amino acid sequence of gshA50;
(B) A protein containing an amino acid sequence containing substitutions, deletions, insertions, and/or additions of 1 to 30 amino acid residues in the amino acid sequence of gshA50 and having γ-Glutamate-cysteine ligase activity; and (C ) A protein containing an amino acid sequence having 90% or more identity to the entire amino acid sequence of gshA50 and having γ-Glutamate-cysteine ligase activity;
and,
The method, wherein said Glutathione synthetase is selected from the group consisting of:
(D) Protein containing the amino acid sequence of gshB ^M165F ;
(E) Substitutions, deletions, and insertions of 1 to 30 amino acid residues in the amino acid sequence of gshB ^M165F ;
(F) A protein that contains an amino acid sequence that has 90% or more identity to the entire amino acid sequence of gshB ^M165F and has Glutathione synthetase activity. .